1. Field of the Invention
This invention is in the fields of cell biology and cellular and molecular pharmacology.
2. Description of the Prior Art
It is known that many molecules of a wide variety are not transported, or are poorly transported, into living cells. Macromolecules, for example, such as proteins, nucleic acids, and polysaccharides, are not suited for diffusion or active transport through cell membranes simply because of their size. In order to allow such macromolecules to pass into cells, cell membranes form small vesicles which migrate from the periphery to the interior of the cell, a process known as pinocytosis. This form of transport is generally less efficient, however, than the diffusion or active transport of smaller molecules, and thus, the cellular uptake of macromolecules is limited.
The most common reason why many small molecules are excluded or poorly transported into cells is their ionic charge. The mere presence of either negatively or positively charged groups severely limits cellular uptake of small molecules, such as nucleotides, nucleotide analogues, cofactors and a number of drugs.
While the two reasons discussed above are the main factors which limit cellular uptake of molecules, there are undoubtedly other reasons as well. Because of these limitations in the cellular uptake of certain molecules, there has been a large amount of research directed to overcoming or obviating inadequate cellular transport of such molecules.
One method previously suggested by Ryser involves the use of cationic polymers, i.e., macromolecules which bear a sequence of positive charges. In this method, it was found that cellular uptake of some molecules could be improved by the simple presence in the experimental medium of such cationic polymers, especially homopolymers of positively charged amino acids such as poly-L-lysines, poly-D-lysines and poly-L-ornithines. See Ryser, H. J.-P., "Uptake of Protein by Mammalian Cells: An Underdeveloped Area", Science, 159, 390-6 (1968); and Ryser, H. J.-P., "Transport of Macromolecules, Especially Proteins Into Mammalian Cells, Proc. Fourth Internat. Congress on Pharmacology (1970); and, Ryser, H. J.-P., "Poly(amino acids) as enhancers in the cellular uptake of macromolecules, in `Peptides, Polypeptides and Proteins`", Proceedings of the Rehovot Symposium on Polyamino Acids, Polypeptides and Proteins and their Biological Implications, May 1974, E. R. Blout, F. A. Bovey, M. Goodman and N. Lotan, Eds, John Wiley and Sons, Inc., New York, pp 617-628 (1974). It has also been shown that protoplasts prepared from mesophyll of Nicotiana tabacum can be infected by adding purified tobacco mosaic virus particles to a protoplast suspension in the presence of poly-L-ornithine whereas infection does not occur if poly-L-ornithine is not present. See Takebe, I. and Otsuki, Y., "Infection of Tobacco Mesophyll Protoplasts by Tobacco Mosaic Virus," Proc. N.A.S., 64, pp 843-8 (1969). The presence of strongly positively charged proteins, such as histones, also increased cellular uptake of albumin. See Ryser, H. J.-P. and Hancock, R., Science, 150, pp 501-3 (1965).
While this prior technique did improve cellular uptake of some macromolecules, it suffered from a number of deficiencies. For example, only minimal enhancement was found for some proteins, e.g., horseradish peroxidase. Also, the cationic polymers form, at most, reversible complexes with the molecule to be transported and may interact at random with other molecules in the medium which means that the enhancement lacks specificity and reproducibility. Further, it was found that enhancement required using cationic polymers of relatively high molecular weight, and that, for example, cationic polymers with molecular weights around 6000 were practically ineffective. See Ryser, H. J.-P., "A Membrane Effect of Basic Polymers Dependent on Molecular Size", Nature, Lond., 215, pp 934-6 (1967). In summary, this technique provided some enhancement of cellular uptake for some macromolecules, but even in these cases enhancement was only modest, non-selective, variable and required polymers of large molecular size.
Other researchers have covalently linked enzymes to polymers for purposes other than cellular transport. Goldstein et al., in U.S. Pat. No. 4,013,511, for example, describe a method for insolubilizing or immobilizing enzymes by covalently bonding the enzymes to anionic or cationic resins. In this method, polymeric resins are formed by reacting ethylene-maleic anhydride copolymer (EMA) and a suitable diamine, such as hydrazine, p,p'-diaminophenyl methane or a primary aliphatic diamine such as 2,6-diaminohexane. Such resins are anionic, but can be made cationic by reacting them with N,N-dimethyl-1,3-propanediamine (DMPA) in the presence of an activating agent, such as dicylohexylcarbodiimide (DCC). Both the anionic and cationic polymeric resins can be covalently coupled to biologically active proteins such as enzymes.
Goldstein et al. ('511) point out that immobilized enzyme derivatives serve as specific easily removable catalysts that can be used repeatedly in columns and in batch reactors. The invention described in U.S. Pat. No. 4,013,511 appears to be a continuation of earlier work by the same researchers which are generally directed to insolubilizing enzymes by reacting them with polymers. See, for example, U.S. Pat. Nos. 3,627,640; 3,650,900; 3,650,901; and 3,706,633. All of these patents contain a description of various techniques which can be used to bond enzymes to polymers. Another patent, namely U.S. Pat. No. 3,374,112, issued to Katchalski et al., discloses the covalent bonding of enzymes to a water insoluble copolymer of L-leucine and p-aminophenyl-DL-alanine, for a similar purpose.
It has also been reported that cationized ferritin can be formed by carbodiimide coupling of horse spleen ferritin to a diamine, namely, N,N-dimethyl-1,3-propanediamine. Cationized ferritin was proposed by these researchers as a tracer molecule for the detection of negatively charged groups on the surface of red blood cells. See Danon, D., Goldstein, L., Marikovsky, Y. and Skutelsky, E., "Use of Cationized Ferritin as a Label of Negative Charges on Cell Surfaces," J. Ultrastructure Res., 38, pp 500-512 (1972); and Grinnell, F., Tobleman, M. Q., and Hackenbrock, C. R., "The Distribution and Mobility of Anionic Sites on the Surfaces of Baby Hamster Kidney Cells," J. Cell Biol., 66, 470 (1975).
A low density lipoprotein was cationized using the Danon et al. technique and shown to accumulate in human fibroblasts. Since low density lipoprotein carries cholesterol into the cells, it was noted that the cationized form of the low density lipoprotein provided an model system for the study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester. See Basu, S. K., Anderson, G. W., Goldstein, J. L., and Brown, M. S., "Metabolism of Cationized Lipoproteins by Human Fibroblasts", J. Cell Biology, 74, pp 119-135 (1977). This method of cationizing a protein has several weaknesses as a method to enhance cellular uptake. For example, the diamine used is not digested by intracellular proteolytic enzymes and thus may accumulate in cells and cause cytotoxic effects. Further, adequate cationization requires attachment of a large number of diamine molecules to carboxyl groups of proteins. Thus, it has been reported that 70% of all of the carboxyl groups of LDL have been modified by this procedure. Such drastic modification would be expected to destroy the biological activity of most functional proteins. Additionally, the diamine used is not suitable as a carrier to enhance the cell penetration of small molecules.
Recent work has been directed to covalently bonding drugs, including anti-cancer drugs, to immunoglobulins or immunoglobulin derivatives for the purpose of directing drugs to cells bearing specific antigens. U.S. Pat. No. 4,046,722, for example, describes an immunoglobulin specific for antigens on the surface of cells to be killed, to which 1-10 polymer carrier molecules are covalently bonded, the polymer carriers having themselves about 5-500 molecules of a cytotoxic drug covalently bonded to them. These polymer carriers have molecular weights of 5000-500,000 and free carboxyl, amino or cycloimidocarbonate groups so that cytotoxic drugs containing amino or carboxyl groups can be covalently bonded thereto. Poly(amino acids), including polylysine, polyaspartic acid, polyarginine, etc., are described as potentially useful polymer carriers for this purpose.
Similar attempts to increase drug selectivity by attaching the drug to antibodies are described in British Patent Specification 1,446,536 and South African patent application 76-2966. These patents disclose techniques for covalently bonding anti-cancer drugs to antibodies or antigen-binding fragments of antibodies specific for tumor antigens.
Still further work involving covalently bonding a potential anti-cancer drug to a polymeric carrier is described in AACR Abstract 454, Proceedings of AACR and ASCO, p 114 (1978). In this work, 6-aminonicotinamide was covalently linked to poly-L-lysine to lower the central nervous toxicity of this drug.